Glutaraldehyde-fixed bioprostheses

ABSTRACT

Methods for treating glutaraldehyde-fixed collagenous tissues to mitigate there propensity for subsequent calcification and to improve durability. Collagenous tissues which have been harvested and cross-linked by glutaraldehyde are exposed to a carboxyl activating agent to convert the free carboxyl (COOH) groups of the collagen molecules to activated carboxyl moieties (e.g., o-acylisourea). Thereafter, the collagenous tissue is exposed to a compound capable of reacting with the activated carboxyl moieties (e.g., o-acylisourea) to form non-carboxyl side groups. Monofunctional and multifunctional amines are examples of compounds which may be utilized to react with the activated carboxyl moieties to form such non-carboxyl side groups. Thereafter, the collagenous tissue is again exposed to glutaraldehyde. If the non-carboxyl side groups have functional amino groups (NH 2 ), such additional exposure to glutaraldehyde will result in additional glutaraldehyde cross-linking of the collagen molecules and resultant improvement of durability.

This application is a division of application Ser. No. 08/688,352 filedJul. 30, 1996 now U.S. Pat. No. 5,782,931.

FIELD OF THE INVENTION

The present invention relates generally to methods of manufacturingbioprosthetic devices, and more particularly to a method for mitigatingcalcification and improving durability of glutaraldehyde-fixedbioprosthetic devices.

BACKGROUND OF THE INVENTION

The prior art has included numerous methods for chemically "fixing"(i.e., tanning) biological tissues. Such chemical fixing of thebiological tissues is often used as a means of preserving such tissuesso that they may be used as, or incorporated into, bioprosthetic deviceswhich are implanted in or attached to a patient's body. Examples offixed biological tissues which have heretofore been utilized asbioprostheses include cardiac valves, blood vessels, skin, dura mater,pericardium, ligaments and tendons. These tissues typically containconnective tissue matrices which act as the supportive framework of thetissues. The cellular parenchyma of each tissue is disposed within andsupported by it's connective tissue matrix.

Collagen and elastin are two substances which make up the connectivetissue framework of most biological tissues. The pliability or rigidityof each biological tissue is largely determined by the relative amountsof collagen and elastin present within the tissue and/or by the physicalstructure and confirmation of the connective tissue frame work.

Each Collagen molecule consists of three (3) polypeptide chains whichare intertwined in a coiled helical confirmation. Chemical fixatives(i.e., tanning agents) used to preserve collagenous biological tissuesgenerally form chemical cross-linkages between the amino groups on thepolypeptide chains within a given collagen molecules, or betweenadjacent collagen molecules.

The chemical cross-linkages formed between polypeptide chains within asingle collagen molecule are termed "intramolecular", while thecross-linkages formed between polypeptide chains of different collagenmolecules are termed "intermolecular".

Chemical fixative agents which have been utilized to cross-linkcollagenous biological tissues include; formaldehyde, glutaraldehyde,dialdehyde starch, hexamethylene diisocyanate and certain polyepoxycompounds.

In particular, glutaraldehyde has proven to be a suitable agent forfixing various biological tissues used for subsequent surgicalimplantation. Indeed, glutaraldehyde has become widely used as achemical fixative for many commercially available bioprostheses, suchas; porcine bioprosthetic heart valves (i.e., the Carpentier-Edwardssstented porcine bioprosthesis; Baxter Healthcare Corporation; EdwardsCVS Division, Irvine, Calif. 92714-5686), bovine pericardial heart valveprostheses (e.g., Carpentier-Edwards SPericardial Bioprosthesis, BaxterHealthcare Corporation, Edwards CVS Division; Irvine, Calif. 92714-5686)and stentless porcine aortic prostheses (e.g., Edwardss PRIMAM StentlessAortic Bioprosthesis, Baxter Edwards AG, Spierstrasse 5, GH6048, Horn,Switzerland).

One problem associated with the implantation of bioprosthetic materialsis that collagen and elastin typically contained in these materials tendto undergo calcification. Such calcification can result in undesirablestiffening or degradation of the bioprosthesis. Both intrinsic andextrinsic calcification are known to occur in fixed collagenousbioprostheses, although the exact mechanism(s) by which suchcalcification occurs is unknown.

Clinical experience and experimental data has taught thatglutaraldehyde-fixed collagenous bioprostheses may tend to calcifysooner than bioprostheses which have been fixed by other nonaldehydefixative agents. Such accelerated calcification of glutaraldehyde-fixedbioprostheses has been reported to occur most predominantly in pediatricpatients. (Carpentier et al., Continuing Improvements in ValvularBioprostheses, J. Thorac Cardiovasc. Surg. 83:27-42, 1982.) Suchaccelerated calcification is undesirable in that it may lead todeterioration and/or failure of the implanted bioprostheses. In view ofthis propensity for accelerated calcification of glutaraldehyde-fixedbioprostheses in young patients, surgeons typically opt to implantmechanical heart valves or homografts (if available) into pediatric orrelatively young patients (i.e., patients under 65 years of age), ratherthan glutaraldehyde-fixed bioprosthetic valves. However, patients whoreceive mechanical valve implants require ongoing treatment withanticoagulant medications, which can be associated with increased riskof hemorrhage. Also, homografts are of limited availability and maycarry pathogens which can result in infection.

The factors which determine the rate at which glutaraldehyde-fixedbioprosthetic grafts undergo calcification have not been fullyelucidated. However, factors which are thought to influence the rate ofcalcification include:

a) patient's age;

b) existing metabolic disorders (i.e., hypercalcemia, diabetes, etc.);

c) dietary factors;

d) race;

e) infection;

f) parenteral calcium administration;

g) dehydration;

h) distortion/mechanical factors;

i) inadequate coagulation therapy during initial period followingsurgical implantation; and

j) host tissue responses.

Many investigators have attempted to discover ways of mitigating the insitu calcification of glutaraldehyde-fixed bioprostheses. Included amongthese calcification mitigating techniques are the methods described inU.S. Pat. No. 4,885,005 (Nashef et al.) entitled Surfactant Treatment ofImplantable Biological Tissue To Inhibit Calcification; U.S. Pat. No.4,648,881 (Carpentier et al.) entitled Implantable Biological Tissue andProcess For Preparation Thereof; U.S. Pat. No. 4,976,733 (Girardot)entitled Prevention of Prosthesis Calcification; U.S. Pat, No. 4,120,649(Schechter) entitled Transplants; U.S. Pat. No. 5,002,2566 (Carpentier)entitled Calcification Mitigation of Bioprosthetic Implants; EP 103947A2(Pollock et al.) entitled Method For Inhibiting Mineralization ofNatural Tissue During Implantation and WO 84/01879 (Nashef et al.)entitled Surfactant Treatment of Implantable Biological Tissue toInhibit Calcification; and, in Yi, D., Liu, W., Yang, J., Wang, B.,Dong, G., and Tan, H.; Study of Calcification Mechanism andAnti-calcification On Cardiac Bioprostheses Pgs. 17-22, Proceedings ofChinese Tissue Valve Conference, Beijing, China, June 1995.

There remains a need in the art for the development of new methods forinhibiting or mitigating calcification of glutaraldehyde-fixedbiological tissues.

SUMMARY OF THE INVENTION

The present invention provides methods for treating glutaraldehydecross-linked tissues which contain collagen and/or elastin so as tomitigate the propensity for subsequent calcification of such tissues, byreplacing at least some of the carboxyl groups present on the collagenand/or elastin molecules with non-carboxyl side groups to therebyeliminate the sites whereby calcium may become chemically or physicallyattached to the protein (i.e., collagen, elastin) molecules. Thereafter,the bioprosthesis may be again immersed in or exposed to glutaraldehyde.If the non-carboxyl side groups formed on the collagen and/or elastinmolecules include glutaraldehyde-reactive groups. (e.g., NH₂ groups),the subsequent exposure to glutaraldehyde will result in the formationof additional glutaraldehyde cross-linkages between saidglutaraldehyde-reacting groups.

In accordance with the present invention, there is provided a methodwhich generally comprises the steps of:

a) providing a collagenous bioprosthesis which has been cross-linkedwith glutaraldehyde;

b) reacting at least some of the carboxyl groups present on collagenmolecules of the bioprosthesis with a carboxyl activating agent toconvert at least some of the carboxyl groups into activated carboxylmoieties;

c) reacting a carboxyl-free compound with said activated carboxylmoieties, thereby forming carboxyl-free side groups on the collagenmolecules of the bioprosthesis.

Additionally, this method may further comprise the additional step of:

d) contacting the bioprosthesis with glutaraldehyde.

The activated carboxyl moieties formed in step d of the above-recitedmethod will typically be o-acylisourea groups of molecular formula CO--.

The bioprosthesis provided in step a of the method may comprise any typeof collagenous tissue such as, heart valves, segments of blood vessel,segments of aortic root having an aortic valve positioned therewithin,pericardium, ligaments, tendons, skin, etc. These collagenous tissuesmay be harvested from any suitable source, and in many instances may beporcine or bovine in origin.

The carboxyl activating agent utilized in step b of the above-summarizedmethod causes the carboxyl (COOH) groups which are present on thecollagen molecules to be converted to activated carboxyl moieties (e.g.,o-acylisourea), which will react with amino groups. Examples of carboxylactivator compounds which may be utilized for this purpose include thefollowing: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride(EDC); dihexylcarbodiimide (DCC);1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide (EAC). Inmany applications 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) is the preferred carboxyl-activating agent.

In step c of the method, a non-carboxyl compound, such as an amine, isreacted with the activated carboxyl moieties (e.g., o-acylisourea)formed in step b, to form non-carboxyl side groups on the collagenmolecules, in place of the previously existing carboxyl (COOH) groups.Due to the relation instability of the activated carboxyl moiety (e.g.,o-acylisourea), it is typically desirable to perform step c (reactionwith non-carboxyl compound) immediately after completion of step b(formation of the activated carboxyl moieties (e.g., o-acylisourea)).Thus, the carboxy-activating agent used in step b and the non-carboxylreactant compound used in step c may desirably be combined in a singlesolution in which the collagenous tissue may be immersed. Thenon-carboxyl side groups formed in step c of the method have lesspropensity for calcification than did the previously-present carboxyl(COOH) side groups of the collagen molecules. Amines are one type ofnon-carboxyl compound which may be reacted with the activated carboxylmoieties (e.g., o-acylisourea), to form the desired non-carboxyl sidegroups on the collagen molecules. When an amine compound is used forthis purpose, the non-carboxyl side groups formed thereby will be boundto the activated carboxyl moieties (e.g., o-acylisourea) by way of amidelinkages therewith. Either monofunctional or multi-functional amines maybe used for this purpose. When monofunctional amines are used for thispurpose, the only functional amino group will be utilized in forming theamide band and the resultant non-carboxyl side groups formed therebywill be free of any remaining amine functionalities. On the other hand,if multifunctional amine compounds are used for this purpose, only onefunctional amino group will be used, in most instances, in forming theamide bond and the resultant non-carboxyl side groups will contain oneor more remaining functional amino groups.

In optional step d of the method, the bioprosthesis may again beimmersed in or otherwise contacted with glutaraldehyde. If thenon-carboxyl side groups formed on the collagen molecules are free offunctional amino groups, this additional exposure to glutaraldehyde willnot result in further cross-linking of the collagen molecules due to theabsence of functional amine bonding sites with which the glutaraldehydemay react. However, if the non-carboxyl side groups formed on thecollagen molecules do contain functional amino groups, this furtherexposure to glutaraldehyde will result in the formation of additionalglutaraldehyde cross-linkages between such remaining free amino groups.

The method of the present invention will also serve to replace thecarboxyl (COOH) groups of the elastin molecules present in thebioprosthesis, with the same non-carboxyl side groups as describedhereabove with respect to the collagen molecules. It will be appreciatedthat, although the invention is described herein as being directed tocollagen molecules in collagenous bioprostheses, most such will alsocontain varying amounts of elastin, and the chemical effects of thepresent invention described herein as affecting the collagen moleculeswill also affect the elastin molecules, due to similarities in thechemical structure of elastin to that of collagen.

Further objects and advantages of the above-summarized invention willbecome apparent to those skilled in the art upon reading of the detaileddescription of preferred embodiment set forth herebelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram of a first preferred embodiment of the methodof the present invention.

FIG. 2 is a schematic diagram of the chemical reactions which occur inthe first preferred embodiment of the method of the present inventionshown in the flow diagram of FIG. 1.

FIG. 3 is a flow diagram of a second preferred embodiment of the methodof the present invention.

FIG. 4 is a schematic diagram showing the chemical reactions which occurin the second preferred embodiment of the method of the presentinvention shown in the flow diagram of FIG. 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following detailed description and the accompanying drawings areprovided for purposes of describing and illustrating certain presentlypreferred embodiments of the invention only, and are not intended tolimit the scope of the invention in any way.

Two (2) embodiments of the invention are shown in the accompanying FIGS.1-4, and described in detail herebelow. Specifically, FIGS. 1-2 aredirected to a first preferred embodiment, while FIGS. 3-4 are directedto a second preferred embodiment.

i. First Preferred Embodiment

Referring to the showings of FIGS. 1-2, the first preferred embodimentof the present invention provides a method for glutaraldehydecross-linking of a collagenous bioprosthesis (Steps 1-2) followed bysubsequent treatment to mitigate it's propensity for subsequentcalcification, and to increase it's durability. In this first embodimentof the invention, the naturally occurring carboxyl groups of thecollagen molecules are replaced (in Steps 4-5 of the method) bynon-carboxyl amide-bound side groups having functional amino groups atthe terminal ends thereof. Thereafter, subsequent exposure toglutaraldehyde (Step 6) results in the formation of additionalglutaraldehyde cross-linkages between the free amine functionalities ofthe non-carboxyl side groups. The formation of such additionalglutaraldehyde cross linkages results in a modification of the physicalproperties of the bioprosthesis, and tends to improve the long-termdurability thereof.

With reference to the flow diagram of FIG. 1, the method of this firstpreferred embodiment comprises the following steps:

Step I: Harvesting and Processing a Collagenous Tissue.

A suitable collagenous tissue is harvested from a mammal, and istrimmed, cleaned and prepared in accordance with standard technique.

Step II: Glutaraldehyde Fixation

In the second step of this method, the previously-prepared collagenoustissue is immersed in 0.1-1.0% glutaraldehyde solution for 30 min. to 2weeks at 4° C.-25° C. to effect glutaraldehyde cross linking betweenfree amino groups located on the collagen molecules of the collagenoustissue.

Step III: Rinsing

After the collagenous tissue is removed from the glutaraldehydesolution, it is rinsed with a suitable phosphate-free rinsing solution,such as 0.9% NaCl solution or HEPES buffer saline. This rinsing removesresidual glutaraldehyde solution from the collagenous tissue. It isdesirable that the rinsing solution be free of phosphates because thepresence of residual phosphates on the collagenous tissue can shortenthe half-life or impair the stability of the carbodimide compound(s)used in the following step IV (described herebelow).

Step IV: Carboxyl Activation and Formation of Non-Carboxyl Side GroupsHaving ANLine Functionality

In this fourth step of the method, the previously glutaraldehyde-fixedcollagenous tissue is immersed in a solution of 1%1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC) and 1% ethylenediamine 2 HCl (EDA) at a pH of 4.5 to 5.0, to a) convert the carboxyl(COOH) groups of the collagen molecules to activated carboxyl moieties(e.g., o-acylisourea), and b) to form amide-bound, non-carboxyl sidegroups on the collagen molecules. The non-carboxyl side groups containfree functional amino groups on the terminal ends thereof.

Step V: Rinsing

After the collagenous tissue is removed from the EDC/EDA solution, it isrinsed with a suitable rinsing solution, such as phosphate bufferedsaline. This rinsing removes residual EDC and EDA from the collagenoustissue.

Step VI: Further Glutaraldehyde Treatment

In this sixth step of the method, the collagenous tissue is immersed in0.1-1.0% glutaraldehyde solution at ambient temperature, until the timeof surgical implantation of the bioprosthesis or subsequentmanufacturing steps (e.g., cutting and mounting on stents or otherframework). This final immersion in glutaraldehyde solution serves tomaintain the sterility of the graft until time of use or furthermanufacturing steps. Furthermore, this final glutaraldehyde treatmentresults in the creation of additional glutaraldehyde cross-linkagesbetween the collagen molecules, as explained more fully herebelow and asshown in detail in FIG. 2.

FIG. 2 provides a schematic showing of the specific chemical reactionswhich take place in Steps IV and VI of the above-summarized firstembodiment of the method. With reference to FIG. 2, it will beappreciated that the ethylene diamine (EDA) used in Step IV of themethod is a straight-chain aliphatic hydrocarbon having terminal amine(NHi) groups located at both ends of the molecule. One of these terminalamine (NH₂) groups reacts with the activated carboxyl moiety (e.g.,o-acylisourea) to form an amide linkage therewith, while the otherterminal amine (NH₂) group remains unreacted and available forsubsequent cross-linking by glutaraldehyde.

Also as shown in FIG. 2, the final exposure of the collagenous tissue toglutaraldehyde in Step VI of the method results in the formation ofadditional glutaraldehyde cross-linkages between the free terminal amine(NH₂) groups which remain on the non-carboxyl side groups of thecollagen molecules.

Thus, the first preferred embodiment of the invention shown in FIGS. 1-2provides not only for replacement of the carboxyl (COOH) side groups ofthe collagen molecules with non-carboxyl side groups having mitigatedpropensity for calcification, but also provides for the formation ofadditional glutaraldehyde cross-linkages which effect the overallcross-linked density and long-term durability of the bioprosthesis.

ii. Second Embodiment

A second preferred embodiment of the invention is shown in FIGS. 3-4.

In this second preferred embodiment of the invention, a monofunctionalamine (propyl amine) is used in Step IV of the method, rather than thedifunctional amine (ethylene diamine) of the above-described firstembodiment. The single amine (NH₂) group on the monofunctional propylamine molecule reacts with the activated carboxyl moiety (e.g.,o-acylisourea) to form an amide linkage therewith. Thus, the resultantcarboxyl-free side group contains no remaining functional amine (NH₂)groups. In this regard, the replacement of the carboxyl (COOH) groups ofthe collagen molecules with the carboxyl-free side groups serves tomitigate the propensity of the bioprosthesis for subsequentcalcification, but the absence of remaining functional amine (NH₂)groups on the carboxyl-free side groups created in Step IV of the methodprevents the collagen molecules from undergoing further glutaraldehydecross-linking during the final exposure to glutaraldehyde.

With reference to the flow diagram of FIG. 3, this second preferredembodiment of the invention is a bioprosthesis preparation method whichcomprises the steps of:

Step I: Harvesting and Processing a Collagenous Tissue.

A suitable collagenous tissue is harvested from a mammal, and istrimmed, cleaned and prepared in accordance with standard technique.

Step II: Glutaraldehyde Fixation

In the second step of this method, the previously-prepared collagenoustissue is immersed in 0.1-1.0% glutaraldehyde solution for 30 min. to 2weeks at 4°-25° C. to effect glutaraldehyde cross linking between freeamino groups located on the collagen molecules of the collagenoustissue.

Step III: Rinsing

After the collagenous tissue is removed from the glutaraldehydesolution, it is rinsed with a suitable phosphate-free rinsing solution,such as 0.9% NaCl solution or HEPES buffer saline. This rinsing removesresidual glutaraldehyde solution from the collagenous tissue. It isdesirable that the rinsing solution be free of phosphates because thepresence of residual phosphates on the collagenous tissue can shortenthe half life or impair the stability of the carbodimide compound(s)used in the following step IV (described herebelow).

Step IV: Carboxyl Activation and Attachment of non-carboxyl Side Groups

In this fourth step of the method, the collagenous tissue is immersed ina solution of 1% 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl(EDC) and 1% propyl amine HCl (PA) for a period of 1 to 10 hours at 4°to 25° C./at a pH of 4.5 to 5.0. This results in a) conversion of thecarboxyl groups (COOH) present on the collagen molecules to activatedcarboxyl moieties (e.g., o-acylisourea) and b) amide bonding of thepropyl amine (PA) molecules with the activated carboxyl moieties (e.g.,o-acylisourea). Thus, the carboxyl groups (COOH) of the collagen chainsare replaced by carboxyl-free side groups. These carboxyl-free sidegroups are devoid of any remaining functional amine (NH₂) groups.

Step V: Rinsing

After the collagenous tissue has been removed from the EDC/PA solution,it is rinsed with a suitable rinsing solution such as phosphate bufferedsaline. This rinsing removes residual EDC and PA from the collagenoustissue.

Step VI: Final Glutaraldehyde Treatment/Sterilization

In this sixth step of the method, the collagenous tissue is immersed in0.1-1.0% glutaraldehyde solution at ambient temperature until time ofsurgical implantation of the bioprosthesis or subsequent manufacturingsteps (e.g., cutting and mounting on stents or other framework). Thisresults in maintained sterilization of the collagenous tissue until timeof use or further manufacturing steps. However, as described more fullyherebelow, this additional glutaraldehyde exposure does not result inthe formation of additional glutaraldehyde cross-linkages because thenon-carboxyl side groups formed on the collagen molecules in Step IV ofthe method are devoid of functional amino groups which could act asbinding sites for the glutaraldehyde.

FIG. 4 is a schematic showing of the chemistry of Steps 4 and 6 of thesecond embodiment of the method shown in the flow diagram of FIG. 3.

With reference to FIG. 4, the1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) converts thecarboxyl (COOH) groups of the collagen chain to activated carboxylmoieties (e.g., o-acylisourea). The single amine (NH₂) groups of thepropyl amine (PA) molecules then react with the activated carboxylmoieties (e.g., o-acylisourea) to form amide linkages therewith. Thisresults in the formation of non-carboxyl side groups which are devoid ofany remaining functional amine (NH₂) groups.

As further shown in FIG. 4, the final exposure of the collagenous tissueto glutaraldehyde in Step VI serves to maintain sterility of thecollagenous tissue, but does not cause further cross-linking of thecollagen molecules due to the absence of functional amine (NH₂) sites onthe non-carboxyl side groups formed by the propyl amine (PA).

Thus, this second embodiment of the method of the present inventiondiffers from the above-described first embodiment in that no furtherglutaraldehyde cross-linking occurs during the glutaraldehyde exposureof Step VI. In this regard, the cross-link density of the collagenoustissue remains unaffected by the treatment method of the secondembodiment, although the propensity for calcification of the collagenoustissue is significantly decreased due to the replacement of theendogenous carboxyl groups (COOH) with non-carboxyl, amide-bound groups,as shown.

It will be appreciated that the invention has been described hereabovewith reference to certain preferred embodiments only. No effort has beenmade to exhaustively describe all possible embodiments in which theinvention may be practiced. It is intended, however that all reasonablemodifications, additions, deletions and variations of theabove-described preferred embodiments be included within the scope ofthe following claims.

What is claimed is:
 1. A glutaraldehyde-fixed bioprosthesis manufacturedby the method comprising the steps of:a) providing a collagen-containingtissue; b) contacting said tissue with glutaraldehyde to form aglutaraldehyde cross-linked tissue; c) rinsing the tissue to removeresidual glutaraldehyde such that the tissue is activated carboxylmoieties on the tissue free of exogenous compounds that would deter thesubsequent formation of activated carboxyl moieties on the tissue: d)contacting the glutaraldehyde cross-linked tissue with a carboxylactivator compound capable of converting at least some of the carboxylgroups present on collagen molecules of the tissue to activated carboxylmoieties capable of reacting with amino groups; e)contacting the tissuewith a compound that will react with the activated carboxyl moieties toform noncarboxyl side groups on the collagen molecules; and, thereafter;f) again contacting said tissue with glutaraldehyde.
 2. Thebioprosthesis of claim 14 wherein the collagenous tissue provided instep a is selected from the group consisting of:heart valves; segmentsof blood vessel; segments of aortic root having an aortic valvepositioned therewithin; pericardium; ligaments; tendons; and, skin. 3.The bioprosthesis of claim 1 wherein the tissue is of porcine origin. 4.The bioprosthesis of claim 1 wherein the tissue is bovine in origin. 5.The bioprosthesis of claim 1 wherein the compound contacted with thetissue in step e is a carboxyl-free monoamine.
 6. The bioprosthesis ofclaim 5 wherein the carboxyl-free monoamine is aliphatic.
 7. Thebioprosthesis of claim 6 wherein the carboxyl-free aliphatic monoamineis propyl amine.
 8. The bioprosthesis of claim 1 wherein the compoundcontacted with the tissue in step e is a carboxyl-free diamine.
 9. Thebioprosthesis of claim 8 wherein the carboxyl-free diamine compound isaliphatic.
 10. The bioprosthesis of claim 9 wherein the carboxyl-free,aliphatic diamine compound is ethylene diamine.
 11. The bioprosthesis ofclaim 10, wherein the compound used in step e contains at least twofunctional amino groups such that at least one functional amino groupwill remain on each amide-bound side group formed on the collagenmolecules in step e, and wherein:the subsequent contacting of the tissuewith glutaraldehyde in step f of the preparation method results in theformation of additional glutaraldehyde cross-linkages between the freeamino groups on the amide-bound side groups of the collagen moleculeswithin the tissue.
 12. The bioprosthesis of claim 1 wherein the carboxylactivator compound used in step c of the preparation method is acarbodiimide.
 13. The bioprosthesis of claim 12 wherein saidcarbodiimide is selected from the group consistingof:1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC);dihexylcarbodiimide (DCC);1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide (EAC).
 14. Aglutaraldehyde-fixed collagenous bioprosthesis prepared by a methodcomprising the steps of:a) providing a collagenous bioprosthesis whichhas been cross-linked with glutaraldehyde and is free of exogenouscompounds that would deter the subsequent formation of activatedcarboxyl moieties on the bioprosthesis; b) reacting at least some of thecarboxyl groups present on collagen molecules of the bioprosthesis witha carboxyl activating agent to convert at least some of the carboxylgroups into activated carboxyl moieties; and, c) reacting acarboxyl-free compound with said activated carboxyl moieties, therebyforming carboxyl-free side groups on the collagen molecules of thebioprosthesis.
 15. The bioprosthesis of claim 14 wherein the carboxylactivating compound is a carbodiimide.
 16. The bioprosthesis of claim 15wherein the carbodiimide is selected from the group consistingof:1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC);dihexylcarbodiimide (DCC); and,1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide (EAC). 17.The bioprosthesis of claim 14 wherein the carboxyl-free compound isselected from the group consisting of:amines; monofunctional amines;multifunctional amines; aliphatic monofunctional amines; aliphaticmultifunctional amines; aliphatic diamines; propyl amine; ethylenediamine.
 18. The bioprosthesis of claim 14 wherein the method by whichthe bioprosthesis is prepared further comprises the step of:d)contacting the bioprosthesis with glutaraldehyde.
 19. The bioprosthesisof claim 18 wherein the carboxyl-free compound used in step c is anamine which has at least two functional amino groups such that onefunctional amino group reacts with the activated carboxyl moiety to forman amide linkage therewith and at least one other functional amino groupwill remain available for subsequent reaction with glutaraldehyde, andwherein the further exposure to glutaraldehyde in step d results in theformation of additional glutaraldehyde cross-linkages between thefunctional amino groups which remain in the carboxyl free side groups ofthe collagen molecules.